The present invention relates to a novel process for the development of an anthocyanin producing callus line in cultures of Panax sikkimensis-Indian Ginseng. More particularly, the developed callus line has characteristic pigmentation, growth kinetics, DNA profile and anthocyanin content.
1. Background
The invention provides a procedure for the development of an anthocyanin producing callus line in cultures of Panax sikkimensis-Indian Ginseng, its growth kinetics and in vitro productivity of anthocyanin in this unique line which also contains ginsenosides that are in high demand in market as important ingredient of health tonics and anti-ageing drug preparation.
The genus Panax (Family Araliaceae) which is commonly called ginseng, has long been known for its saponin (ginsenosides) responsible for anti-ageing, adaptogenic and immunomodulatory activity. We have been engaged in biotechnological studies in American and Indian species of Panax for past several years. While screening cell lines specifically rich in various ginsenoside fractions, from callus cultures of an Indian speciesxe2x80x94P. sikkimensis. we came across with anthocyanin rich cell clusters which have subsequently been cloned through continuous cell-aggregate selection procedure.
2. Prior Art References
Anthocyanins are widely formed in various plant species and are most conspicuous in flower and fruit parts. Because of their low toxicity, anthocyanins have a high potential as a food additive and marker. Therefore, many institutes and food manufacturers are engaged in intensive research to produce these pigments from various plant cell cultures such as Euphorbia milli (Agri.Biol.Chem.53:417-423, 1989); Callistephus chinensis (Pl.Cell Rep.5:435-438,1986); Vitis vinifera (Biotech.Agri.Forest.Vol.24,Med. PI. V,ed.Y. P. S.Bajaj,pp 373-386,1993); Srawbeny (J.Sci.Food Agric.66:381-388,1994); Perilla frutescens (J.Ferment. Bioeng. 76: 530-531,1993); Aralia cordata (PI. Cell Tiss. Org. Cul. 36: 21-26,1994) etc. Anthocyanins, however, usually accumulate only in small amounts in cultured plant cells and their production generally requires light irradiation. One of the highest reports of anthocyanin production has been in Glehnia littoralis (Phytochemistry 48: 279-282, 1998) callus cultures where as high as 14.2% g xe2x88x921 D.Wt. anthocyanin production has been obtained. In many cases, anthocyanins produced in vitro are of cyanidin type which are biochemically more primitive than those of the original plant where methylated antrhocyanidins are present (Rev. Can. Biol. Exp.42:13-18, 1983). Seven different anthocyanidins were detected:cyanidine (17 spp.), delphinidin (5 spp.) malvinidine (4 spp.) petunidin (3 spp.), pelargonidin (2 spp.) and one example for both peonidin and hirsutidin.
Although in vitro anthocyanin production has been reported in many other genera mostly in callus/suspension cultures (Phytochemistry 29: 2153-2158,1990, Biotechnol. Agric. Forest. Vol. 24; Ed. Bajaj pp 373-386, 1993), to the best of our knowledge there has been no report of its production in the genus Panax Panax is probably the sole source of an important group of secondary metabolites namely ginsenosides which are used as adaptogens. The root extract/powder of this important plant is marketed in the form of tonics, teas, chewing gums, face creams (for rejuvenating skin) etc. Occurrence of anthocyanin in the same callus along with ginsenosides will further add to its market potential. The coloured extract with ginsenosides can be used in candy""s, cakes, pastries, cold drinks etc.
The main object of the present invention is to devise a procedure for the development of an anthocyanin producing callus line in cultures of Indian ginseng Panax sikkimensis with characteristic pigmentation, growth kinetics, DNA profile and anthocyanin content. To the best of our knowledge so far, nobody has reported occurrence of anthocyanin in callus cultures of any Panax species especially P. sikkimensis. 
Another object of the present invention is to provide a novel P. sikkimensis callus line capable of producing high quantity of anthocyanin
Yet another object of the present invention is to study the growth kinetics in relation to anthocyanin production in callus cultures of P. sikkimensis. 
Still another object of the present inventions is to extract ginsenosides from anthocyanmin producing callus line.
Still another object of the present invention is to identify the physical conditions for increased anthocyanin production.
Still another object of the present invention is to characterise the anthocyanin producing line at the molecular level.
Still another object of the present invention is to characterize the anthocyanin pigment. Still another object of the present invention is simultaneous recovery of anthocyanin and ginsenoside.
To meet the above objects and others, the present invention provides a novel Panax sikkimensis (an Indian species of ginseng) callus line which produces anthocyanin, said line comprising:
(a) Characteristic pink-purple pigmentation,
(b) Growth index of about 221-450 in about 50-80 days of culture period,
(c) Yield of anthocyanin (2-3 mg/gf.wt.) from the callus in about 40-60 days under light conditions (continuous light), and
(d) Characteristic DNA profile wherein the lanes 1,2,3,4, 5,6,7,8,9,10,11 and 12 in each gel (upper gel for wild line and lower gel for anthocyanin producing line) represent the PCR amplified fragments produced by the respective template megabase genomic DNA with primers 5xe2x80x2 CTG ATG CAT C, 3xe2x80x2,5xe2x80x2 TGG TCA CTG A 3xe2x80x2, 5xe2x80x2 AGG GGT CTT G 3xe2x80x2,5xe2x80x2 GAA ACG GGT G 3xe2x80x2,5xe2x80x2 AGG GGT CTT G 3xe2x80x2, 5xe2x80x2 GCG TAA CGC C 3xe2x80x2,5xe2x80x2 CAG CAC CCA C 3xe2x80x2,5xe2x80x2 GTT GCG ATC C 3xe2x80x2, 5xe2x80x2 CAG GCC CTT C 3xe2x80x2,5xe2x80x2 CGC AGT ACT C 3xe2x80x2,5xe2x80x2 GTC CTA CTC G 3xe2x80x2,5xe2x80x2 CTA CAC AGG C 3xe2x80x2 and 5xe2x80x2 GTC CTT AGC G 3xe2x80x2, respectively. Lane M shows the standard size markers of a ladder at 100 bp intervals from 1000 bp downwards.
The present invention also provides a method for the development of an anthocyanin producing line of P. sikkimensis from root explants wherein the said method comprising the steps of:
(a) establishment of aseptic cultures of root explants of P. sikkimensis on modified Murashige and Skoogs medium (Medium I) to obtain callus,
(b) transferring the primary calli to medium II for optimal growth along with anthocyanin production,
(c) maintaining and multiplying the callus for over 3 years by regular subculturing in medium II at every 4-6 weeks under dark and/or light cycle, temperature 28xc2x13xc2x0 C., 70-80% relative humidity,
(d) isolating the anthocyanin producing callus line by selectively subculturing the cell culture which exhibited the presence of anthocyanin for atleast 5-8 subsequent subcultures, and
(e) proliferating the enriched callus line by using medium II and incubating the callus cultures under specified light conditions (16 hrs. light/8 hrs. dark or 24 hrs. light).
a) To the best of our knowledge, the present invention reports for the first time a procedure for the development of anthocyanin producing callus line in P. sikkimensis with appreciably high anthocyanin content(2.16% F. Wt.) in addition to the characteristic ginsenosides (0.9-1.2% F. Wt). The procedure outlines the protocol for induction of callus from excised root pieces of P. sikkimensis. a callus culture multiplication and maintenance medium (nutrient+growth supplements) and incubation environment (physical conditions) that support sustained growth and anthocyanin production over more than 3 years tested so far.
b) The invention has resulted in obtaining the pigment anthocyanin as well as ginsenosides from the same callus tissue.
c) The invention has resulted in bench level identification of parameters such as light conditions (light-dark cycle, continuous light and complete darkness) and harvesting schedule, hormone concentration and specification etc. for anthocyanin production.
d) The developed line has a morphologically distinguishable feature, the accompanying photograph, (sheet 1, FIG. 1) depict the characteristic dark pink purple pigmentation in the callus cells of the isolated line in comparison to the wild counterpart. The morphological appearance is as nearly true as is reasonably possible to make the same in colored illustration of this character and
e) The developed line has characteristic DNA profile (Sheet 1, FIG. 2).
In a preferred embodiment of the process the roots are cut into 4-7 mm small explants; are sterilized by treatment with 1% cetabelon (v/v) for a period of about 5-15 minutes, then with 70% (V/v) ethanol for 30 sec., followed by treatment with 0.1% HgCl2 (w/v) for about 1-2 min. The basal medium in step (a) is modified Murashige and Skoog""s (1962) medium supplemented with 200 mg/l myoinositol, 10 mg/l each of thiamine hydrochloride and pyridoxine hydrochloride and 5 mg/l of nicotinic acid (Medium I), and medium II used in step (b) is obtained by addition of 2,4-dichlorophenoxyacetic acid (0.1-2.0 mg/l) and Kn (0.1-0.5 mg/l), that are preferably added to the basal medium for optimal callus growth.